DESCRIPTION: (Applicant's Abstract) ATP-sensitive potassium channels (KATP channels) couple metabolism to membrane electrical activity. In pancreatic beta-cells KATP channels alter the membrane potential in response to changes in the ATP/ADP ratio driven by glucose metabolism. To understand how KATP channels function, we cloned and reconstituted them, characterized two sets of human genes that encode their subunits, sulfonylurea receptors (SURs) and inward rectifiers (KIR6.x) respectively, and showed that mutations in SUR1 and KIR6.2 cause a recessive form of familial hyperinsulinism. The subunit functions are tightly integrated, both are required for assembly, regulation and surface expression of the heteromeric (SUR1/KIR6.2)4 channel; subunits expressed alone are retained in the ER. C-terminal truncation of KIR6.2 removes an ER retention signal allowing surface expression of a K+ channel with low sensitivity to ATP, highly modified kinetics and none of the pharmacological properties of KATP channels. Co-expression with SUR1 largely restores the native channel properties. Co-expression of SUR1 with N-terminally truncated KIR6.2 gives a channel with greatly prolonged bursts and reduced ATP sensitivity. beta-cell (SUR1) and cardiac (SUR2A) channel isoforms exhibit different kinetics and ATP sensitivity. SUR chimeras have been used to identify two regions that specify the kinetic and ATP sensitivity differences. These results begin to define a web or network of interactions that place an unidentified ATP-binding site on KIR6.2, two intracellular segments of SUR, and the N-terminus of KIR6.2 in close proximity to the mouth of the channel. Disruption of this network of interactions can affect ATP-inhibitory gating in complex ways. In this competitive renewal we propose to define the interactions within this network using peptide display, mammalian two hybrid, and epitope tagging techniques to identify the peptide segments involved. Specifically we hypothesize there are interactions between: 1) Transmembrane domain 1 (TMD1) of KIR6.2 and the TMDs of SUR, 2) the N-terminus of KIR6.2 and a segment within the first set of TMDs of SUR, 3) the C-terminus of SUR and the last 36 amino acids of KIR6.2, and 4) the last 52 amino acids of SUR and the C-terminal cytoplasmic domain of KIR6.2.